New data on the zoogeography of Aphanius sophiae (Teleostei: Cyprinodontidae) in the Central Zagros (Southwest Iran)

Accepted Manuscript Title: New data on the zoogeography of Aphanius sophiae (Teleostei: Cyprinodontidae) in the Central Zagros (Southwest Iran) Author: Zeinab Gholami Hamid Reza Esmaeili Bettina Reichenbacher PII: DOI: Reference:

S0075-9511(14)00081-4 http://dx.doi.org/doi:10.1016/j.limno.2014.12.002 LIMNO 25433

To appear in: Received date: Revised date: Accepted date:

23-7-2014 1-12-2014 1-12-2014

Please cite this article as: Gholami, Z., Esmaeili, H.R., Reichenbacher, B.,New data on the zoogeography of Aphanius sophiae (Teleostei: Cyprinodontidae) in the Central Zagros (Southwest Iran), Limnologica (2014), http://dx.doi.org/10.1016/j.limno.2014.12.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

New data on the zoogeography of Aphanius sophiae (Teleostei: Cyprinodontidae) in the Central Zagros (Southwest Iran) Zeinab Gholamia,*, Hamid Reza Esmaeilib, Bettina Reichenbachera a

Department of Earth and Environmental Sciences, Palaeontology and Geobiology and GeoBio-

Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran

ip t

Center LMU, Ludwig-Maximilians-University, Munich, Germany b

cr

*Corresponding author. Zeinab Gholami ([email protected]/[email protected])

us

Tel: office: +49 (0) 89 21806618

an

Fax: +49 (0) 89 21806601

Ac ce p

te

d

M

Running headline: Aphanius sophiae in the Central Zagros (SW-Iran)

23

Page 1 of 32

Abstract The clade of the Iranian freshwater Aphanius species from endorheic and exorheic drainage basins contains three subclades, of which the A. sophiae subclade with seven species is the most specious one. Recently, two previously not known populations of Aphanius were discovered in two isolated basins; one in the Arjan Wetland (Helleh subbasin), and the other in the Semirom spring (Karun

ip t

Basin), both are located in the Central Zagros Mountains (SW Iran). The objective of this study is to investigate their taxonomic status, to elucidate their phylogenetic relationships and to contribute to future conservation strategies and habitat management of the freshwater species of Aphanius in

cr

Iran. Methods include analysis of genetic data based on mtDNA (cyt b), combined with meristics, morphometrics, scale sizes (J-indices) and otolith data. The results based on cyt b clearly indicate

us

that two species are present in the Arjan Wetland, one is closely related to A. sophiae (currently thought to be restricted to the Kor Basin), the other represents A. shirini (previously only known

an

from its type locality Paselari spring). However, significant phenotypic differences are not present between these two species. The second population from the Semirom spring is sister to A. sophiae (Kor Basin) according to cyt b data, but differs significantly from this species with regard to the

M

phenotype. The presence of A. shirini in the Arjan Wetland is most likely be explained by manmade introduction because of the recent droughts. The similarity of the two species present in the

d

Arjan Wetland may be due to phenotypic plasticity, but also hybridization could have played a role. The isolation of populations of A. sophiae is discussed in the context of the active geological history

te

and climate change, and it is likely that their divergence happened in the Early or Middle Holocene (c. 11,700–4,000 y. ago). The presence of A. sophiae in the Helleh subbasin and Karun Basin

Ac ce p

extends the currently known zoogeographic range of this species, which previously has only been reported from the Kor Basin. Such knowledge is important for future conservation strategies and habitat management.

Key words: Aphanius; evolution; conservation; genetic differentiation; Holocene; aridity

1. Introduction 24

Page 2 of 32

Species of the killifish genus Aphanius Nardo, 1827 (Teleostei, Cyprinodontiformes) are widely distributed in coastal habitats and freshwater to euryhaline inland waterbodies throughout the Mediterranean region and the Middle and Near East (e.g. Wildekamp, 1993; Ferrito et al., 2013; Kottelat and Freyhof, 2007). Iran and central Anatolia keep the highest species diversity of Aphanius, and speciation events have largely been linked to vicariance and changes in drainage

ip t

systems due to the geological history (Hrbek et al., 2002, 2006; Hrbek and Meyer, 2003; Esmaeili et al., 2012; Teimori et al., 2012a; Gholami et al., 2014; Esmaeili et al., 2014a, b).

To date, 14 Aphanius species have been described from the Alburz (NE-Iran) and Central

cr

Zagros (SW-Iran) Mountains of the Iranian plateau. They are representatives of three large clades based on previously published phylogenetic trees derived from molecular data:

us

(i) The A. dispar clade is distributed in the Persian Gulf, Gulf of Oman, and the Red Sea and Arabian Seas. It involves four species, i.e. A. dispar, A. ginaonis, A. furcatus, and A. sirhani (Hrbek

an

and Meyer, 2003; Teimori et al., 2014).

(ii) The A. mento clade is a large clade comprising many distinct populations in the Eastern Mediterranean and Anatolia (Wildekamp et al., 1999; Hrbek et al., 2002; Hrbek and Meyer, 2003).

M

Aphanius mento was recorded from southern Iran near the border to Iraq (Wildekamp, 1993), but its existence in this area has not been confirmed in later studies.

d

(iii) The Inland and Inland-related Aphanius species (IIRAS) clade comprises ten species (A. vladykovi, A. darabensis, A. shirini, A. isfahanensis, A. farsicus, A. arakensis, A. kavirensis, A.

te

mesopotamicus, A. pluristriatus and A. sophiae) (Hrbek et al., 2006; Gholami et al., 2014; Esmaeili et al., 2014b) (Fig. 1). According to Esmaeili et al. (2014b), the IIRAS clade contains three

Ac ce p

subclades (A. vladykovi-, A. shirini- and A. sophiae subclade) and represents an “old” evolutionary group that had diverged in the Late Miocene and Early Pliocene (A. vladykovi and A. shirini subclades and A. isfahanensis of the A. sophiae subclade) and a “young” evolutionary group that may have diverged during the Late Pleistocene to Middle Holocene (A. sophiae subclade, but without A. isfahanensis).

The A. sophiae subclade is the most species’ rich group of the IIRAS clade. A remarkable feature of the “young” Aphanius species in the A. sophiae subclade is that they are superficially very similar and often not clearly distinguishable by external characters alone (Hrbek et al., 2006; Teimori et al., 2012b; Esmaeili et al., 2014b). However, differences in cytochrome b and also between the otoliths clearly separate all species of this subclade (Teimori et al., 2012b; Gholami et al., 2014; Esmaeili et al., 2014b). In addition, the species of the A. sophiae subclade show widely separated distribution areas with no hydrological networks or connectivity in between, which 25

Page 3 of 32

additionally supports their interpretation as distinctive species (Teimori et al., 2012b; Esmaeili et al., 2014b). In September 2012, two previously not known populations of Aphanius were discovered in two isolated basins; one in the Arjan Wetland (Helleh subbasin), and the other in the Semirom spring in the uppermost reaches of the Karun Basin, both are located in the Central Zagros Mountains (Figs.

ip t

1 and 2). The objective of this study is to investigate the taxonomic status of these populations, to elucidate their phylogenetic relationships in order to understand their evolutionary history, and thus to contribute to future conservation strategies and habitat management of the inland and inland-

us

1.1. Study regions

cr

related species and populations of Aphanius in Iran.

The Persian Gulf drainage basin of southern Iran comprises the Helleh, Zohreh and Mond

an

subbasins (Coad, 2014, Fig. 1). The Arjan Wetland, where one of the new populations was found, is part of the Helleh subbasin (28°42' to 30°20' N, 50°35' to 52°10' E), which is a large freshwater marsh system covering 10,000 km2 including the Arjan Wetland, Parishan Lake, the Khesht and

M

Komaj Plains and two main rivers, i.e. the Shapour and Dalaki Rivers (Fig. 2A). The rivers flow together near Borazjan city, where they create the Helleh River and eventually drain into the

d

Persian Gulf. Elevation ranges around 500–800 m in the southeastern Khesht plain, while the highest mountains are located near to the Arjan Wetland, with 2850–3020 m above sea level

te

(Chahbarfi Mountains, see Shiati, 1991; Teimori, 2006; Monavari and Fard, 2011). The Arjan Wetland is an outstanding freshwater system recorded on the International wetland list since 1975

Ac ce p

(Darvish Sefat, 2007; Mosleh et al., 2013); its size varies annually depending on rainfall and it holds a great diversity of plants and animals (Darvish Sefat, 2007; Mosleh et al., 2013). The Karun River Basin (30°17' to 33°49' N, 48°15' to 52°30' E), from where the second new populations comes, comprises an area of 61,000 km2 and extends over a length of 720 km; elevations range from 8 to 4367 m (Nourani and Mano, 2007; Noori et al., 2010) (Fig. 1). The Karun River is the most important river with the largest mean discharge in Iran; it drains to the Arvand River (or Shatt al Arab) and Bahmanshir River, which both flow to the northwestern Persian Gulf (Fig. 2B).

2. Materials and Methods 2.1. Material 26

Page 4 of 32

All specimens are deposited in the Zoological Museum of Shiraz University, Collection of Biology Department (ZM-CBSU), Iran. Thirty individuals of Aphanius from the Arjan Wetland were used for both morphological and molecular studies (Tables 1 and 2). Twenty-two individuals from the Semirom spring were used for morphological investigations, and seven individuals out of these 22 specimens as well as seven additional samples from this site were used for the molecular

ip t

study (Tables 1 and 2). Specimens were collected using dip nets, transferred to 5% ethanol for 10 min to avoid shrinkage, and then preserved in 96% ethanol (formalin solution was not used, as it

cr

dissolves otoliths and inhibits molecular studies). 2.2. Comparative material

us

In addition to the newly collected fishes, 14 individuals of Aphanius sophiae (Heckel, in: Russegger, 1846) were included originating from the Maloosjan spring, which is located near to the

an

type locality of this species in the Kor Basin, and nine specimens of A. shirini Gholami, Esmaeili, Erpenbeck and Reichenbacher, 2014 from its type locality, i.e. the Paselari spring of the Khosroshirin River in the upper reaches of the Kor Basin (same specimens as used in Gholami et

M

al., 2014) (Fig. 1, Table 1). Sequences of further Aphanius species and of outgroups were obtained from the NCBI GenBank (http://www.ncbi.nlm.nih.gov) and included in the molecular analysis

te

2.3. Molecular analyses

d

(Table 2).

A small piece of dorsal muscle of each alcohol preserved fish was removed in sterile condition

Ac ce p

and placed in 96-well Eppendorf PCR plates until further processing. Total genomic DNA was purified by extraction of tissues with high concentrations of Guanidinium Thiocyanate and passage of the extracts through fiberglass membranes (AcroPrep 1 µM glass fiber; PALL 5051) as described by Vargas et al. (2012). The entire cytochrome b gene was amplified via PCR using the primers (forward:

L14724

(5’-GTGACTTGAAAAACCACCGTTG-3’;

reverse:

H15915

(5’-

CAACGATCTCCGGTTTAGAAGAC-3’) (Schmidt and Gold, 1993; Perdices et al., 2001). Amplification was performed under the following temperature regime: initial 92oC for 3 min, 34 cycles at 92oC for 1 min, 53oC for 90 sec and 72oC for 3 min, and a final extension step at 72oC for 4 min. PCR products were purified with the PEG (Polyethylene glycol) method (Rosenthal et al., 1993). Cleaned DNA was sequenced in a cycle sequencing reaction using BigDye 3.1 chemistry (Applied Biosystems, Munich, Germany) according to the protocol provided by the manufacturer. The same forward and reverse primers were used as for PCR, and the following temperature program was employed: initial 96oC for 1 min, 30 cycles at 96oC for 15s, 52oC for 10 min, 60oC for 27

Page 5 of 32

2 and 30 min. Sequencing was performed on an ABI 3730 automated sequencer in the Genomic Sequencing Unit, Department of Biology, LMU, Munich. Sequences were assembled, edited and aligned with MUSCLE (Edgar, 2004) under default setting as incorporated in Geneious version 6.4 (Drummond et al., 2012). The achieved cytochrome b gene sequences for the here studied Aphanius populations are deposited in GenBank under accession numbers KJ135737–KJ135780 (Table 2).

ip t

Maximum likelihood reconstructions with PhyML were estimated using SeaView version 4 (Guindon et al., 2010; Gouy et al., 2010). The GTR + G option yielded the best-fitting model of nucleotide substitution, as computed using the program jModelTest 2.1.1 under the Akaike

cr

Information Criterion (Darriba et al., 2012).

Phylogenetic relationships based on Bayesian inference (BI) was estimated using MrBayes 3.2 total 2,000,000

us

(Ronquist et al., 2012) with two runs of four Markov Chain Monte Carlo (MCMC) chains over a generations under the most generalized model (GTR+G+I), because

an

overparametrization obviously does not negatively affect Bayesian analyses (Huelsenbeck and Ranala, 2004). Chains were stopped after the standard deviation of split frequencies fell below 0.01, and the likelihood values of sampled trees from both runs reached a stationary distribution. The first

M

25% of trees before were disregarded as "burnin". The evolutionary divergence over sequence pairs between studied Aphanius species were calculated using the Kimura 2-parameter model (Kimura,

d

1980).

te

2.4. Morphological and statistical analyses

Nine meristic characters were counted under a stereomicroscope. They include the numbers of

Ac ce p

the branched and unbranched dorsal, anal, pectoral and pelvic fin rays, the numbers of gill rakers of the first branchial arch, and the numbers of caudal peduncle scales, lateral scales series, predorsal scales and white flank bars (Table 3). Following Holcik (1989), 15 morphometric parameters of each fish individual were measured using a vernier caliper adjusted to the nearest 0.5 mm and the measurements were standardized in relation to the standard length (SL) (see Lahnsteiner and Jagsch, 2005). In total, 14 morphometric variables were calculated and served as input for the statistical analyses (Table 3).

Four scales from the 3rd or 4th row below the dorsal fin from the left side of each fish were removed, then mounted between two microscope slides, and length and width of scales were measured to the nearest 0.1 mm using an oculometer attached to a stereomicroscope. For each individual, the measurements of scale length and scale width were averaged to obtain a single length and a single width value and relative length and width of scales in relation to SL (= J-index) were calculated (Esmaeili, 2001; Gholami et al., 2013) (Table 3). 28

Page 6 of 32

For otolith analysis, fish skulls were opened dorsally, and left and right saccular otoliths were removed. Otoliths were cleaned from organic remains with 1% potassium hydroxide solution for 3– 4 hours, rinsed in distilled water for 4–5 hours, and then washed several times with distilled water (Reichenbacher et al., 2007; Gholami et al., 2014). Otolith morphology was studied using a stereomicroscope and scanning electron microscopy (SEM) (LEO 1430 VP). For otolith

ip t

morphometrics, digital images from left otoliths were captured using a Leica DFC 295 camera and three angles and eight linear distances of each left otolith were measured using the Leica Image Access Software (IMAGIC 1000, Imagic Bildverarbeitung AG, Glattbrugg, Switzerland). Ten

cr

otolith variables were calculated as input for the statistical analyses (Reichenbacher et al., 2007) (Table 3). Due to the ontogenetic variation, we used only otoliths from fish with SL ≥ 20 mm for

us

the morphometric otolith analyses (see Reichenbacher et al., 2009).

Datasets derived from meristics, morphometrics, scales and otoliths were merged for male and

an

females in the statistical analyses because otherwise sample sizes would have been too low. Statistical analyses were carried out using PASW 21.00 (SPSS Inc, 2013). Univariate analysis of variance (ANOVA, with Duncan’s post hoc test, p < 0.05) was used to test the significance of

M

phenotypic differences between populations. The t-test (p < 0.05) was applied to test whether variables showed significant differences between two groups. The canonical discriminant analysis

d

(CDA) was used for multivariate analyses in order to show the classification success between the

3. Results

te

groups.

Ac ce p

3.1. Phylogenetic relationships

The PhyML and Bayesian likelihood based on the Cyt b gene were largely congruent. Both phylogenetic trees indicate that the individuals from the Arjan Wetland belong to two lineages. One of which (represented by 20 individuals) is sister to A. sophiae from the type area in the Kor Basin (Maloosjan) + the individuals from the Semirom spring (Fig. 3); it clearly represents a population of A. sophiae and is termed A. sophiae (Arjan) in the following. The second lineage (represented by ten individuals) forms a monophyletic unit with A. shirini (Paselari) and clearly represents a population of A. shirini; it is termed A. shirini (Arjan) in the following. Aphanius sophiae (Arjan) and A. shirini (Arjan) are separated from each other by 58 fixed molecular apomorphies (44 transitions and 14 transversions). The individuals of A. sophiae (Arjan) have eight fixed molecular apomorphies (five transitions and three transversions) compared to A. sophiae (Maloosjan). Aphanius sophiae (Maloosjan) shows 61 fixed molecular apomorphies (46 29

Page 7 of 32

transitions and 15 transversions) compared to A. shirini (Paselari), and 58 fixed molecular apomorphies (45 transitions and 13 transversions) compared to A. shirini (Arjan). The individuals of Aphanius from the Semirom spring form a monophyletic group, which is sister to A. sophiae (Maloosjan), and, together with this population, is sister to A. sophiae (Arjan) (Fig. 3). This lineage clearly represents another population of A. sophiae; it is termed A. sophiae

ip t

(Semirom) in the following. The individuals of A. sophiae (Semirom) varied in nine fixed molecular apomorphies (seven transitions and two transversions) from A. sophiae (Maloosjan).

Bootstrap values less than 70% and short branch lengths indicate that the phylogenetic

cr

relationships between the three lineages of A. sophiae (Arjan, Semirom, Maloosjan) are poorly resolved (Fig. 3). Estimation of evolutionary divergence based on the Kimura 2-parameter model

us

suggests low genetic distances between the three populations of A. sophiae (Arjan, Semirom,

an

Maloosjan) (0.006, 0.008; see Appendix).

3.2. Morphological data of Aphanius sophiae (Arjan) and A. shirini (Arjan) 3.2.1. Comparisons of phenotypes

M

Aphanius sophiae (Arjan) vs. A. shirini (Arjan). No significant differences were found based on meristics, morphometrics and J-indices (t-test, p < 0.05) (Table 3 and Fig. 4).

d

Aphanius sophiae (Arjan) vs. A. sophiae (Maloosjan). The following characters were found to be different (t-test p < 0.05): (i) six meristic characters, i.e. numbers of dorsal fin rays (12.5±1.1 vs.

te

13.3±0.8), anal fin rays (11.3±0.9 vs. 12.7±0.4), pelvic fin rays (5.5±0.5 vs. 6.0±0.0), gill rakers (9.8±0.8 vs. 10.8±0.9), predorsal scales (14.9±1.1 vs. 15.7±1.4), and white flank bars (13.1±1.4 vs.

Ac ce p

14.5±1.0); (ii) five morphometric variables (given in % of SL), i.e. head lengths (28.8±1.5 vs. 30.3±1.4), predorsal distances (60.9±2.2 vs. 62.9±1.2), postdorsal distances (47.5±6.6 vs. 43.7±1.6), maximum body depths (28.5±3.4 vs. 33.0±2.2), and caudal peduncle lengths (24.1±1.6 vs. 22.3±1.5); (iii) J-indices, i.e. scale lengths in % of SL (2.43±0.4 vs. 4.30±0.3) and scale widths in % of SL (2.78±0.5 vs. 5.3±0.4) (Table 3 and Fig. 4). Aphanius shirini (Arjan) vs. A. shirini (Paselari spring). The following characters were found to be different (t-test p < 0.05): (i) six meristic characters, i.e. numbers of dorsal fin rays (13.5±1.0 vs. 10.8±0.9), anal fin rays (11.5±0.6 vs. 10.5±0.6), lateral scales series (27±0.8 vs. 28.5±0.6), caudal peduncle scales (9.0±0.0 vs. 10.3±0.9), predorsal scales (15.3±0.9 vs. 21.8±0.5), and white flank bars (11.0±1.0 vs. 8.3±1.2); (ii) six morphometric variables (given in % of SL), i.e. predorsal distances (60.5±1.0 vs. 64.7±0.4), maximum body depths (26.6±0.9 vs. 29.4±2.2), dorsal fin depths (17.8±0.8 vs. 14.9±0.9) and lengths (16.3±0.2 vs. 12.5±0.5), prepelvic (54.6±3.1 vs. 57.3±1.9) and 30

Page 8 of 32

prepectoral (33.1±2.0 vs. 31.6±0.9) distances; (iii) J-indices, i.e. scale lengths in % of SL (2.29±0.6 vs. 3.4±0.3) and scale widths in % of SL (2.50±0.7 vs. 3.8±0.2) (Table 3 and Fig. 4). 3.2.2 Otolith data and comparisons Otoliths of A. sophiae (Arjan) are triangular to rounded-trapezoid, and characterized by a wide,

ip t

V-shaped excisura, and a well-developed slightly pointed rostrum and antirostrum; the rostrum is usually longer than the antirostrum. The dorsal margin can be slightly crenulated; the posterior margin is rounded or angular (Fig. 5). Otoliths of A. sophiae (Maloosjan) are rounded-triangular to

cr

rounded-trapezoid, with a U- or V-shaped excisura, and similar to those of A. sophiae (Arjan). They show a well-developed, pointed rostrum and a prominent and rounded antirostrum; the rostrum is

us

clearly longer than the antirostrum. The dorsal margin is curved; the posterior rim is steep and a prominent posteroventral edge is present in most specimens (Fig. 5). No significant differences

an

between A. sophiae (Arjan) and A. sophiae (Maloosjan) were found based on the otolith morphometry (Table 3 and Fig. 5).

Otoliths of A. shirini (Arjan) are similar to those of A. sophiae (Arjan), but the rostrum length

M

is twice of the antirostrum length in some individuals. Some of the specimens are rounded-trapezoid in shape and then similar to the otoliths of A. shirini (Paselari), whereas others are triangular in

d

shape like those of A. sophiae (Arjan) (Fig. 5). The similarity between the otoliths of A. shirini (Arjan) and A. sophiae (Arjan) is additionally indicated by the otolith morphometry because only

te

the length-height index is significantly different between them (Table 3 and Fig. 5). Otoliths of A. shirini (Paselari) are quadrangular to trapezoid, with a wide excisura. They

Ac ce p

display a short rostrum and antirostrum of almost equal size, the rostrum tip is truncated or rounded, the antirostrum tip rounded or slightly pointed. The dorsal margin is broadly curved and slightly crenulated; the posterior margin is steep and in some specimens crenulated (Fig. 5). Notably, three otolith variables differ significantly between A. shirini (Arjan) and A. shirini (Paselari), i.e. the relative rostrum lengths (19.38±4.3 vs. 11.19±2.5), the relative medial (77.84±7.2 vs. 83.92±1.5) and relative dorsal lengths (74.53±6.6 vs. 83.07±1.8) (Table 3 and Fig. 5). Furthermore, the measurements of the otolith lengths for all individuals that are available in size classes 2–3 indicates that A. shirini (Paselari and Arjan) possess slightly larger otoliths than A. sophiae (Table 4). In all otoliths described above, the sulcus is straight and covered with a structure containing some openings of different sizes (see Fig. 5). The ostium is ovate to rounded and shorter in length than the cauda. In the otoliths of A. sophiae (Maloosjan), the ostium is usually opened to the anterior margin, while in the otoliths of A. shirini (Paselari), the ostium is not opened to the anterior 31

Page 9 of 32

margin. The otoliths of A. shirini and A. sophiae from Arjan can display an ostium that is either opened or not opened (Fig. 5). 3.3. Morphological data of Aphanius sophiae (Semirom and Maloosjan) 3.3.1. Comparisons of phenotypes

ip t

Several significant phenotypic differences appeared between the individuals of A. sophiae from Semirom and Maloosjan with regard to (i) seven meristic characters, i.e. numbers of dorsal fin rays (12.4±0.5 vs. 13.3±0.8), anal fin rays (11.4±0.5 vs. 12.7±0.4), pectoral fin rays (14.9±1.3 vs.

cr

16.0±0.9), pelvic fin rays (5.9±0.4 vs. 6.0±0.0), gill rakers (9.9±1.1 vs. 10.8±0.9), caudal peduncle scales (8.4±0.5 vs. 9.8±0.8), and white flank bars (12.3±1.3 vs. 14.5±1.0); (ii) six morphometric

us

variables (given in % of SL), i.e. postdorsal distances (48.9±2.4 vs. 43.7±1.6), preorbital distances (8.9±1.5 vs. 7.5±0.6), maximum body depths (25.3±1.3 vs. 33.0±2.2), caudal peduncle lengths

an

(24.2±0.7 vs. 22.3±1.5), dorsal fin lengths (16.3±1.4 vs. 18.2±1.4), and pelvic fin lengths (9.0±0.5 vs. 9.7±1.1), and (iii) J-indices, i.e. scale lengths in % of SL (3.21±0.28 vs. 4.30±0.3) and scale

M

widths in % of SL (3.75±0.21 vs. 5.3±0.4) (Table 3 and Fig. 4). 3.3.2 Otolith data and comparisons

d

Otoliths of A. sophiae (Semirom) are rounded-triangular to rounded-quadrangular, with a very wide excisura. The rostrum displays a pointed or rounded tip and is longer or as long as the

te

antirostrum. The dorsal margin shows few crenulations in some specimens and the posterior margin is steep (Fig. 5). The general sulcus morphology is the same as described above, the ostium is

Ac ce p

usually opened to the anterior margin. Only the length-height index is significantly different between the individuals of A. sophiae from Semirom and Maloosjan (0.93±0.03 vs. 1.06±0.09). In addition, the measurements of the otolith lengths for all individuals that are available in size classes 2–3 indicate that otoliths are smallest in A. sophiae (Semirom) (Table 4). 3.4. Comparisons of Aphanius sophiae (Arjan, Maloosjan, Semirom) The studied individuals of A. sophiae come from three nowadays-isolated drainage basins, i.e. the Helleh subbasin (Arjan), the Karun Basin (Semirom) and the Kor Basin (Maloosjan). Comparisons of their meristic characters and morphometric variables have revealed some significant differences between them (ANOVA, Duncan post-hoc test, p < 0.05) (Table 3): The sample from Arjan displays the lowest number of predorsal scales (14.9±1.1 vs. 15.7±1.4 or more) and the lowest value of the predorsal distance (60.9±2.2 vs. 62.9±1.2 or more). The individuals from Semirom depict the lowest numbers of pectoral fin rays (14.9±1.3 vs. 15.9±0.9 or 32

Page 10 of 32

more), caudal peduncle scales (8.4±0.5 vs. 9.2±0.7 or more) and white flank bars (12.3±1.3 vs. 13.1±1.4 or more), and also the lowest value of the maximum body depth (25.3±1.3 vs. 28.5±3.4 or more). The specimens from Maloosjan possess the highest numbers of dorsal fin rays (13.8±1.2 vs. 13.0±0.8 or less), anal fin rays (12.7±0.4 vs. 11.4±0.5 or less), pelvic fin rays (6.1±0.4 vs. 5.5±0.5 or less), gill rakers (10.9±0.6 vs. 10.2±0.6 or less), and white flank bars (14.5±1.0 vs. 13.1±1.4 or

ip t

less), the highest value of the maximum body depth (33.8±2.2 vs. 27.0±2.8 or less), and the lowest values of both the postdorsal distance (42.5±1.7 vs. 46.8±2.0 or more) and the caudal peduncle length (22.4±1.5 vs. 24.2±0.7 or more).

cr

The CDA (jackknifed) based on meristic characters indicates a good classification success (> 71%) for A. sophiae from Arjan and Maloosjan, but not for the specimens from Semirom (Table 5).

us

The CDA (jackknifed) based on morphometric variables achieves a good classification success (> 77%) for A. sophiae from Semirom and Maloosjan, but not for the sample from Arjan (Table 5).

an

When both meristics and morphometrics are used for the CDA (jackknifed), a very good classification success (> 90%) is obtained for A. sophiae from Semirom and Maloosjan, while the classification success is good (75%) for A. sophiae from Arjan (Table 4).

M

Furthermore, J-indices are significantly different between the three studied samples of A. sophiae (ANOVA, Duncan post-hoc test, p < 0.05) and the CDA (jackknifed) based on the J-indices

d

shows a very good classification success for each group (≥ 90%) (Table 5). The largest J-indices are seen in A. sophiae (Maloosjan) (scale length 4.30±0.3; scale width 5.03±0.4), those of A. sophiae

te

(Semirom) are intermediate (3.21±0.28; 3.75±0.21), while J-indices of A. sophiae (Arjan) were small (2.43±0.4; 2.78±0.5) (Table 3).

Ac ce p

Apart from the length-height index of the otoliths of A. sophiae (Semirom) (0.93±0.03 vs. 0.99±0.06 or more), the otolith variables are not significantly different between the three studied samples of A. sophiae (ANOVA, Duncan post-hoc test, p < 0.05). The CDA (jackknifed) based on the otolith variables supports moderate classification success for A. sophiae (Semirom) (69.2%) and low classification success for the remaining groups (Table 5E). 4. Discussion

4.1. Biogeographic history The Iranian plateau faced different active tectonic events during the Late Miocene (5.3–11.6 Ma) and the Pliocene (2.6–5.3 Ma). In addition, a young phase of very intensive Pleistocene tectonics (2.6 Ma–11.700 y. ago) has been described (Stöcklin, 1968; Dercourt et al., 1986; Hatzfeld et al., 2010). The tectonic phases caused new structures of mountain ranges, isolation of multiple areas, migration of barriers and new drainage patterns (Dercourt et al., 1986; Hrbek and 33

Page 11 of 32

Meyer, 2003; Hrbek et al., 2006; Gholami et al., 2014). The most important deformations took place along the Main Zagros Reverse Fault (MZRF) of the North and Central Zagros (Fig. 6A), and also along the Main Recent Fault (MRF) of the North Zagros (Fig. 6A) (Talebian and Jackson, 2004; Agard et al., 2005; Hatzfeld et al., 2010; Mouthereau, 2011). A further important tectonic structure is the Kazerun Fault System (KFS, between 51°E to 54°E), which is separating the North

ip t

Zagros from the Central Zagros (Fig. 6A). The KFS includes several individual faults, i.e. the Kazerun fault consisting of three segments (Dena, Kazerun, Borazjan), and the Kareh-Bas, SabzPushan and Sarvestan faults (Fig. 6B).

cr

The Arjan Wetland is located near to the Kareh-Bas Fault (Fig. 6B), which represents the most active fault of the KFS (see Hatzfeld et al., 2010) since c. 2.8–0.8 Ma (Late Pliocene–Early

us

Holocene) (Authemayou et al., 2009). The Semirom spring is near to the Dena segment of the Kazerun Fault (Fig. 6B), which is active since c. 3 Ma (Late Pliocene) (Authemayou et al., 2009;

an

Hatzfeld et al., 2010). It is therefore possible that the isolation of the A. sophiae populations in the Arjan Wetland and the Semirom spring can be linked to the motion of these faults, which caused interruption of formerly interconnected drainage systems and isolation of populations. The onset of

M

the isolation events could have occurred in the time span of Late Pliocene to Early Holocene (a more precise dating has not been achieved for the tectonic activities). However, in light of the

d

previously assumed “young” age of the A. sophiae subclade (Esmaeili et al., 2014b), and considering the very short branches of the molecular tree for the three studied populations of A.

te

sophiae (Fig. 3), we assume that their splits took place in the Early or Middle Holocene (c. 11,700– 4,000 y. ago). It is possible that not only vicariance due to the geological activity, but also climate

Ac ce p

change and increased aridity in the course of the Holocene (Kehl, 2009) played a role in the isolation and divergence of the populations of A. sophiae. 4.2. Presence of Aphanius shirini in the Arjan Wetland There is no hydrological connection that would allow gene flow between the individuals of A. shirini in the upper reaches of the Kor River Basin (Paselari spring) and those in the Arjan Wetland. However, the Arjan Wetland experienced a severe drought and dried almost completely in the year 2000 and thousands of fishes died (Global Environment Facility, 2004; www.iransabz.org/news/fish2.htm, Reuters, 8 June 2000, taken from Coad, 2014). This was the reason that the Environmental Organisation of the Fars Province (Iran) transferred fish from watery springs in the mountains that still hold fish to the Arjan Wetland (unpublished data of H.R.E). It appears that they used individuals of Aphanius from the Paselari spring (Kor Basin), which at that time were thought to represent a population of A. sophiae, but in fact represented A. shirini (see Gholami et al. 34

Page 12 of 32

2014). Obviously some native fishes had survived the drought of the Arjan Wetland and this is the reason that native A. sophiae now co-occur with the artificially introduced individuals of A. shirini. 4.3. Phenotypic similarity between Aphanius sophiae (Arjan) and A. shirini (Arjan) No phenotypic variation was recognized between A. sophiae (Arjan) and A. shirini (Arjan), but

ip t

the molecular data (cyt b) clearly indicate that they belong to different clades. This means that they do not form a monophyletic clade and thus cannot be considered as representing the same species. It is possible that some of the phenotypic similarities result from phenotypic plasticity as they

cr

shared the same habitat and experienced the same environmental conditions. The reduced size in the predorsal distance and, in the males, also in the postdorsal distance of A. shirini from the Arjan

us

habitat (compared to A. shirini from the large-sized Paselari spring) support this assumption because these characters are related to swimming abilities (see also Gholami et al. in press).

an

However, further characters that differ between the two populations of A. shirini cannot clearly be linked to environmental parameters. It is therefore likely that hybridisation of the two species in the

M

Arjan wetland has caused their high phenotypic similarity (see below). 4.4. Phenotypic variation between Aphanius shirini (Arjan and Paselari)

d

A significant criterium for the discrimination of Aphanius shirini from the type locality (Paselari) is that it has the lowest number of white flank bars among all inland species of Aphanius

te

from Iran (Gholami et al., 2014). The number of white flank bars has also been recognized as taxonomically useful for A. arakensis, which has the highest number of white flank bars among all

Ac ce p

inland species of Aphanius (Teimori et al., 2012b). However, A. shirini (Arjan) displays a higher number of white flank bars as seen in A. shirini from the type locality (11±1.0 vs. 8.3±1.2), but the small sample size for male individuals (n = 3) precludes further discussion whether this is an artefact or a valid trait.

Furthermore, several meristic and morphometric characters were found to differ significantly between the studied individuals of A. shirini from the Arjan Wetland and the Paselari spring (Table 3). Several of these significantly varying characters represent traits that were reported as taxonomically useful at the species level, e.g. for the identification of A. vladykovi (number of lateral scale series, see Coad, 1996) and A. sophiae (maximum body depth and prepelvic distance, see Hrbek et al., 2006; Esmaeili et al., 2014b). Variations in the predorsal distance, and relative rostrum length and relative medial length of otoliths have also been reported to differ significantly between isolated populations of A. dispar (Teimori et al., 2012a). Eventually, differences in numbers of caudal peduncle scales and lateral scale series, caudal peduncle lengths, and relative 35

Page 13 of 32

medial and dorsal lengths of otoliths have been observed in interconnected populations of A. farsicus from habitats with different environmental parameters (Gholami et al., in press). However, isolation cannot account for the high phenotypic variation between A. shirini (Arjan) and A. shirini (Paselari) because the introduction of A. shirini to the Arjan Wetland happened only in the year 2000. Besides environmental parameters, we suppose that hybridization between A. shirini (Arjan)

ip t

and A. sophiae (Arjan) contributes to the observed differences between the individuals of A. shirini from the Arjan Wetlands and the Paselari spring. Such hybridization could explain why color patterns and otoliths of A. shirini (Arjan) and A. sophiae (Arjan) are intermediate in comparison to

cr

A. shirini (Paselari) and A. sophiae (Maloosjan) (see Figs. 4 and 5). The idea that otoliths can trace hybridisation events is supported by the study of Schulz-Mirbach et al. (2008), who found that the

us

otolith contour of the killifish Poecilia formosa, which is a hybrid of P. latipinna and P. mexicana, is intermediate to those of the parental species.

an

Despite of the phenotypic differences, ten meristic and morphometric characters are almost constant between A. shirini from Arjan and Paselari (Table 3). Among them are several characters that have been reported as taxonomically important. Examples are the number of gill rakers

M

(significant for A. pluristriatus vs. A. isfahanensis, A. sophiae, A. farsicus; see Esmaeili et al., 2012), the caudal peduncle length (significant for A. shirini vs. other inland species of Aphanius in

d

Iran; see Gholami et al., 2014), and the pelvic fin length as well as the pectoral fin length (significant for A. darabensis and A. kavirensis, respectively, vs. other inland species of Aphanius in

te

Iran). The same characters were found to remain stable between populations of A. farsicus inhabiting sites with different environmental parameters (see Gholami et al., in press). In addition,

Ac ce p

there are two otolith variables that are not different between A. shirini from Arjan and Paselari, i.e. the length-height index and the height of the rostrum. These otolith variables have been reported to be diagnostic at the species level in previous work (Reichenbacher et al., 2007, 2009; Teimori et al., 2012b; Gholami et al., in press). It can therefore be assumed that the above reported meristic, morphometric and otolith characters are independent from environmental parameters and contain valuable taxonomic information.

4.5. Phenotypic variation between Aphanius sophiae (Arjan, Maloosjan, Semirom) Up to now, several Aphanius species are known that are difficult to identify based on external characters, but are clearly separated based on genetic data. Examples are A. arakensis Teimori et al., 2012b, A. kavirensis Esmaeili et al., 2014b, A. pluristriatus (Jenkins, 2010) and also A. baeticus Doadrio et al., 2002. It is therefore quite surprising to observe high phenotypic variation between the individuals of A. sophiae from Arjan, Semirom and Maloosjan despite of their close 36

Page 14 of 32

relationships. It is possible that the observed variation may be, at least partially, related to the isolation of the populations due to their tectonically induced habitat fragmentation since the Early or Middle Holocene, as discussed above. In addition, the high phenotypic plasticity of individuals of Aphanius, when exposed to different habitats, probably also played a role. For example, a long postdorsal distance and a long caudal peduncle can enhance efficient swimming (see studies on

ip t

Salvelinus fontinalis and S. alpinus by Dynes et al., 1999 and Peres-Neto and Magnan, 2004). Thus, the long postdorsal distance in A. sophiae (Semirom) may be related to improved swimming capability because of the relatively large size of the Semirom spring. In addition, the small caudal

cr

peduncle and postdorsal distance in A. sophiae (Maloosjan) may indicate no demand for proficient swimming because the size of Maloosjan spring is rather small. Furthermore, the relative otolith

us

size is significantly smaller in A. sophiae (Semirom) than in A. sophiae from Arjan and Maloosjan (see Table 4), which may perhaps be related to some differences in water temperature, water depth

an

and diet (see e.g. Lombarte and Lleonart, 1993; Tuset et al., 2003; Mérigot et al., 2007). Another unexpected result was that the relative scale sizes (J-indices) are significantly different between the three populations of A. sophiae because J-indices have been reported as a reliable

M

taxonomic character in previous work, e.g. for the identification of A. sophiae, A. farsicus, A. isfahanenis, A. pluristriatus, A. vladykovi, A. arakensis, A. darabensis and A. kavirensis (Esmaeili

d

et al., 2012, 2014b; Teimori et al., 2012b; Gholami et al., 2013). It appears that differences of Jindices were evolving due to long-time isolation of the studied populations (e.g. since the Early or

te

Middle Holocene), and that J-indices may not be taxonomically useful on the species level in every

Ac ce p

case. 5. Conclusion

(I) A combined analysis of molecular data and morphological datasets can disentangle the complex relationships of closely related Aphanius species in Iran and contributes to the understanding of character evolution in the context of differing environments and/or allopatric divergence. Such studies are also important for the identification of local populations and species, and for their conservation strategy and habitat management. (II) The record of A. shirini in the Arjan Wetland is due to artificial introduction of fish after a severe drought in 2000. Our data clearly show that careful taxonomic studies are necessary before fishes are introduced in order to protect native populations. (III) Populations of A. sophiae are not restricted to the Kor River Basin, as previously thought. They are recorded here for the first time from the isolated Helleh Subbasin and from the upper reaches of the Karun Basin. The isolation of these newly discovered populations of A. sophiae 37

Page 15 of 32

probably took place in the Early or Middle Holocene (c. 11,700–4,000 y. ago) due to the prominent tectonic activity of the Main Zagros Reverse Fault and motion of the Kazerun Fault System in the Central Zagros, and possibly also because of climate change and increased aridity in the course of the Holocene.

ip t

Acknowledgements

Financial support has been provided by DAAD (German Academic Exchange Services) to the

cr

first author. Special thanks go to PD Dr. D. Erpenbeck (LMU, Munich) for his help regarding the phylogenetic interpretations. We also thank Prof. Dr. G. Wörheide (LMU, Munich) for providing

us

access to the Molecular Geo-and Palaeobiology Lab at LMU Munich, Dr. T. Schulz-Mirbach (LMU, Munich) for constructive discussion, Prof. R. Melzer (ZSM, Munich) for providing access to the SEM at the Bavarian State Collection of Zoology, and Dr. F. Soroush (Isfahan University,

an

Iran), Dr. M. Asadpour (Shahid Beheshti University of Tehran, Iran), Dr. I. Bariani (Gorgan University of Agricultural Sciences and Natural Resources, Iran) and Dr. M. Sharifipour (Shahid

M

Chamran University of Ahvaz, Iran) for kindly providing some references for this project. Moreover, we would like to thank R. Zamaniannejad, M. Masoudi, A. Gholamifard, G.

References

te

Semirom spring.

d

Sayyadzadeh, S. Mirghiasi and M. Ghasemian for helping in fish collection from Arjan wetland and

Ac ce p

Agard, P., Omrani, J., Jolivet, L., Mouthereau, F., 2005. Convergence history across Zagros, Iran; constraints from collisional and earlier deformation. Iranian J. Earth Sci. 94, 401–419. Authemayou, C., Bellier, O., Chardon, D., Benedetti, L., Malekzadeh, Z., Claude, C., Angeletti, B., Shabanian, E., Abbassi, M. R., 2009. Quaternary slip-rates of the Kazerun and the Main Recent Faults: active strike-slip partitioning in the Zagros fold-and-thrust belt. Geophys. J. Int. 178, 524–540.

Coad, B.W., 1996. Systematics of the tooth-carp genus Aphanius Nardo, 1827 (Actinopterygii: Cyprinodontidae) in Fars Province, southern Iran. Biol. Bratislava 51, 163–172. Coad, B.W., 2014. Freshwater fishes of Iran. Available from http://www.briancoad.com (accessed June 2014). Darriba, D., Taboada, G., Doallo, R., Posada, D., 2012. "jModelTest 2: more models, new heuristics and parallel computing". Nature Methods 9, 772–772.

38

Page 16 of 32

Darvish Sefat, A.A., 2007. Atlas of protected areas of Iran. Tehran: Tehran University Publication, ISBN: 964039114 . Dercourt, J., Zonenshain, L.P., Ricou, L.E., Le Pichon, X., Knipper, A.L., Grandjaquet, C., Sbortshikov, I.M., Geussant, J., Lepvrier, C., Pechersku, D.H., Boulin, J., Bazhenov, M. L., Lauer, J.P., Biju-Duval, B., 1986. Geological evolution of the Tethys belt from the Atlantic to

ip t

the Pamirs since the Lias. Tectonophysics 123, 241–315. Doadrio I., Carmona J.A., Fernández-Delgado, C., 2002. Morphometric study of the Iberian Aphanius (Actinopterygii, Cyprinodontiformes), with description of a new species. Folia Zool.

cr

51, 67–79.

Drummond, A.J., Ashton, B., Buxton, S., Cheung, M., Cooper, A., Duran, C., Field, M., Heled, J.,

us

Kearse, M., Markowitz, S., Moir, R., Stones-Havas, S., Sturrock, S., Thierer, T., Wilson, A., 2012. Geneious v6.4, Available from http://www.geneious.com.

an

Dynes, J., Magnan, P., Bernatchez, L., Rodriguez, M.A., 1999. Genetic and morphological variation between two forms of lacustrine brook char. J. Fish Biol. 54, 955–972. Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high

M

throughput. Nucleic Acids Res. 32, 1792–1797.

Esmaeili, H.R., 2001. Some aspects of biology of an exotic fish Hypophthalmicthys molitrix (Val.,

d

1844) from Gobindsagar reservoir Himachal paradesh, India. PhD Thesis, Department of Zoology, Panjab University, Chandigarh, 287p.

te

Esmaeili, H.R., Teimori, A., Gholami, Z., Zarei, N., Reichenbacher, B., 2012. Re-validation and redescription of an endemic and endangered species, Aphanius pluristriatus (Jenkins, 1910)

Ac ce p

(Teleostei, Cyprinodontidae), from southern Iran. Zootaxa 3208, 58–67. Esmaeili, H.R., Teimori, A., Sayyadzadeh, G., Masoudi, M., Reichenbacher, B., 2014a. Phylogenetic relationships of the tooth-carp Aphanius (Teleostei: Cyprinodontidae) in the river systems of southern and south-western Iran based on mtDNA sequences. Zool. Middle East 60, 29–38.

Esmaeili, H.R., Teimori, A., Gholami, Z., Reichenbacher, B., 2014b. Two new species of the toothcarps Aphanius (Teleostei: Cyprinodontidae) and the evolutionary history of the Iranian inland and inland-related Aphanius species. Zootaxa 3786, 246–268. Ferrito, V., Pappalardo, A.M., Canapa, A., Barucca, M., Doadrio, I., Olmo, E., Tigano, C., 2013. Mitochondrial phylogeography of the killifish Aphanius fasciatus (Teleostei, Cyprinodontidae) reveals highly divergent Mediterranean populations. Mar. Biol. 160, 3193–3208.

39

Page 17 of 32

Gholami, Z., Teimori, A., Esmaeili, H.R., Schulz-Mirbach, T., Reichenbacher, B., 2013. Scale surface microstructure and scale size in the tooth-carp genus Aphanius (Teleostei, Cyprinodontidae) from endorheic basins in Southwest Iran. Zootaxa 3619, 467–490. Gholami, Z., Esmaeili, H.R., Erpenbeck., Reichenbacher, B., 2014. Phylogenetic analysis of Aphanius from the endorheic Kor River Basin in the Zagros Mountains, South-western Iran

ip t

(Teleostei: Cyprinodontiformes: Cyprinodontidae). J. Zool. Sys. Evol. Res. 52 (2), 130–141. Gholami, Z., Esmaeili, H.R., Erpenbeck, D., Reichenbacher, B., in press. Genetic connectivity and phenotypic plasticity in the cyprinodont Aphanius farsicus from the Maharlu Basin, South-

cr

western Iran. J. Fish Biol.

Global Environment Facility, 2004. Conservation of Iranian Wetlands – PIMS no, 980. Co-

us

operation with Ministry of Energy, Ministry of Agricultural Jihad and Management and Planning Organization. UNDP Project Document. Pp, 201, page 9.

an

Gouy, M., Guindon, S., Gascuel, O., 2010. SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol. Biol. Evol. 27, 221–224. Guindon, S., Dufayard, J.F., Lefort, V., Anisimova, M., Hordijk, W., Gascuel, O., 2010. New

M

Algorithms and Methods to Estimate Maximum-Likelihood Phylogenies: Assessing the Performance of PhyML 3.0. Syst. Biol. 59, 307–21.

d

Hatzfeld, D., Authemayou, C., Vanderbeek, P., Bellier, O., Lave¢, J., Oveisi, B., Tatar, M., Tavakoli, F., Walpersdorf, A., Yamini-fard, F., 2010. The kinematics of the Zagros Mountains

te

(Iran). Geol. Soc. Lond. 330, 19–42.

Holcik, J., 1989. The Freshwater Fishes of Europe. General Introduction to Fishes:

Ac ce p

Acipenceriformes. AULA-Verlag, Wiesbaden, 469 p. Hrbek, T., Küçük, F., Frickey, T.N., Stölting, K., Wildekamp. R.H., Meyer, A., 2002. Molecular phylogeny and historical biogeography of the Aphanius (Pisces, Cyprinodontiformes) species complex of central Anatolia, Turkey. Mol. Phyl. Evol. 25, 125–137. Hrbek, T., Meyer, A., 2003. Closing of the Tethys Sea and the phylogeny of Eurasian killifishes (Cyprinodontiformes: Cyprinodontidae). J. Evol. Biol. 16, 17–36. Hrbek, T., Keivany, Y., Coad, B.W., 2006. New species of Aphanius (Teleostei, Cyprinodontidae) from Isfahan Province of Iran and a reanalysis of other Iranian species. Copeia 2, 244–255. Huelsenbeck, J.P., Ranala, B., 2004. Frequentist properties of Bayesian posterior probabilities of phylogenetic trees under simple and complex substitution models. Sys. Biol. 53, 904–913. Jenkins, J.T., 1910. Notes on fish from India and Persia, with descriptions of new species. 1. On a collection of fishes made by W. T. Blainford in 1872 in Persia and Baluchistan. Record of 40

Page 18 of 32

Indiana Museum 5, 123–128.

Kehl, M., 2009. Quaternary climate change in Iran, the state of knowledge. Erdkunde 63, 1–17. Kimura, M., 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111–120.

ip t

Kottelat, M., Freyhof, J., 2007. Handbook of European Freshwater Fishes. Kottelat, Cornol, Switzerland and Freyhof, Berlin, Germany.

cr

Lahnsteiner, F., Jagsch, A., 2005. Change in phenotype and genotype of Austrain Salmo trutta populations during the last century. Environ. Biol. Fish. 74, 51–65.

us

Lombarte, A., L1eonart, J., 1993. Otolith size changes related with body growth, habitat depth and temperature. Environ. Biol. Fish. 37, 297–306.

Mérigot, B., Letourneur, Y., Lecomte-Finiger, R., 2007. Characterization of local populations of the

an

common sole Solea solea (Pisces, Soleidae) in the NW Mediterranean through otolith morphometrics and shape analysis. Mar. Biol. 151, 997–1008.

M

Monavari, M., Fard, S.M.B., 2011. Application of network method as a tool for integrating biodiversity values in Environmental Impact Assessment. Environmental Monitoring assessment, Springer, 172, 145–156. Doi: 10.1007/s10661–010–1323–9.

d

Mouthereau, F., 2011. Timing of uplift in the Zagros belt/Iranian plateau and accommodation of

te

late Cenozoic Arabia-Eurasia convergence. Geol. Mag. 148, 726–738. Mosleh, L., Asadi, F., Safaian, R., 2013. Investigation on the values of International Parishan

Ac ce p

Lake‘s plants. Int. J. Adv. Biol. Biomed. Res. 1 (10), 1263–1270. Noori, R., Sabahi, M.S., Karbassi, A.R., Baghvand, A., Taati-Zadeh, H., 2010. Multivariate statistical analysis of surface water quality based on correlations and variations in the dataset. Desalination 260, 129–136.

Nourani, V., Mano, A., 2007. Semi-distributed flood runoff model at the subcontinental scale for southwestern Iran. Hydrol. Prog. 21, 3173–3180. Perdices, A., Carmona, J.A., Fernandez-Delgado, C., Doadrio, I., 2001. Nuclear and mitochondrial data reveal high genetic divergence among Atlantic and Mediterranean populations of the Iberian killifish Aphanius iberus (Teleostei, Cyprinodotidae). Heredity 87, 314–324. Peres-Neto, P.R., Magnan, P., 2004. The influence of swimming demand on phenotypic plasticity and morphological integration: a comparison of two polymorphic charr species. Oceanologia 140, 36–45.

41

Page 19 of 32

Reichenbacher, B., Sienknecht, U., Küchenoff, H., Fenske, N., 2007. Combined otolith morphology and morphometry for assessing taxonomy and diversity in fossil and extant killifish (Aphanius, †Prolebias). J. Morphol. 268, 898–915. Reichenbacher, B., Feulner, G.R., Schulz-Mirbach, T., 2009. Geographic Variation in Otolith Morphology

Among

Freshwater

Populations

of

Aphanius

dispar

(Teleostei,

ip t

Cyprinodontiformes) from the Southeastern Arabian Peninsula. J. Morph. 270, 469–484. Ronquist, F., Teslenko, M., Van der Mark, P., Ayres, D., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M.A., Huelsenbeck, J.P., 2012. MrBayes 3.2: Efficient Bayesian Phylogenetic

cr

Inference and Model Choice across a Large Model Space. Syst. Biol. 61, 539–542.

Rosenthal, A., Coutelle, O., Craxton, M., 1993. Large-scale production of DNA sequencing

us

templates by microtitre format PCR. Nucleic Acids Res. 21, 173–174.

Schmidt, T.R., Gold, J.R., 1993. Complete sequence oft he mitochondrial cytochrome b gene in the

an

cherryfin shiner, Lythrurus roseipinnis (Teleostei, Cyprinidae). Copeia 3, 880–883. Schulz-Mirbach, T., Scherb, H., Reichenbacher, B., 2008. Are hybridization and polyploidization phenomena detectable in the fossil record? – A case study on otoliths of a natural hybrid,

M

Poecilia formosa (Teleostei: Poeciliidae). N. Jb. Geol. Paläont. 249, 223–238. Shiati, K., 1991. A regional approach to salinity management in river basins. A case study in

d

southern Iran. Agricultural Water management 19, 27–41. SPSS Inc., 2013. IBM SPSS Statistics for Mac. Version 21.0. Armonk, NY: IBM Corp. 1229–1258.

te

Stöcklin, J., 1968. Structural history and tectonics of Iran: a review. Am. Assoc. Pet. Geol. 52,

Ac ce p

Talebian, M., Jackson, J., 2004. A reappraisal of earthquake focal mechanisms and active shortening in the Zagros Mountains of Iran. Geophys. J. Int. 156, 506–526. Teimori, A., 2006. Biodiversity of Freshwater Fishes in Southern Iran- Fars Province. Master thesis, Shiraz University, Shiraz, 190 p.

Teimori, A., Schulz-Mirbach, T., Esmaeili, H.R., Reichenbacher, B., 2012a. Geographical differentiation of Aphanius dispar (Teleostei: Cyprinodontidae) from Southern Iran. J. Zool. Sys. Evol. Res. 50, 289–304.

Teimori, A., Esmaeili, H.R., Gholami, Z., Zarei, N., Reichenbacher, B., 2012b. Aphanius arakensis, a new species of tooth-carp (Actinopterygii, Cyprinodontidae) from the endorheic Namak Lake basin in Iran. ZooKeys 215, 55–76. Teimori, A., Esmaeili, H.R. Erpenbeck, D., Reichenbacher, B., 2014. A new and unique species of the genus Aphanius (Teleostei: Cyprinodontidae) from Southern Iran: A case of regressive evolution. Zool. Anz. 253 (4), 327–337. 42

Page 20 of 32

Tuset, V.M., Lombarte, A., Gonza´lez, J.A., Pertusa, J.F. Lorentes, M.J., 2003. Comparative morphology of the sagittal otolith in Serranus spp. J. Fish Biol. 63, 1491–1504. Vargas, S., Schuster, A., Sacher, K., Büttner, G., Schätzle, S., Läuchli, B., Hall, K., Hooper, J. N. A., Erpenbeck, D., Wörheide, G., 2012. Barcoding Sponges: An Overview Based on Comprehensive Sampling. PLoS ONE 7, e39345.

ip t

Wildekamp, R.H., 1993. A world of Killies. Atlas of the Oviparous Cyprinodontiform Fishes of the World. The Genera Adamas, Adinia, Aphanius, Aphyoplatys and Aphyosemion. American Killifish Association, Indiana.

cr

Wildekamp, R.H., Kücük, F., Ünlüsayin, M., Neer, W.V., 1999. Species and subspecies of the

Ac ce p

te

d

M

an

us

genus Aphanius Nardo 1827 (Pisces: Cyprinodontidae) in Turkey. Turk. J. Zool. 23, 23–44.

43

Page 21 of 32

Ac

ce

pt

ed

M

an

us

cr

i

Figure

Page 22 of 32

Ac

ce

pt

ed

M

an

us

cr

i

Figure

Page 23 of 32

Ac ce p

te

d

M

an

us

cr

ip t

Figure

Page 24 of 32

Ac

ce

pt

ed

M

an

us

cr

i

Figure

Page 25 of 32

Ac

ce

pt

ed

M

an

us

cr

i

Figure

Page 26 of 32

Ac

ce

pt

ed

M

an

us

cr

i

Figure

Page 27 of 32

Table

Table 1. Details of the samples studied, comparative material was previously used in Gholami et al. (2014). SD, standard deviation; SL, standard length in mm; N, number of specimens. SL range and mean (±SD) Basin/site/altitude/GPS data

New material A. sophiae

ZM-CBSU ZG 100–101, 103–113, 257–261, 263, 266

A. sophiae

301–302, 304–305, 307, 323– 329, 338–339, j809, j869, j872, j886, 888–889, j891, j894 102, 256, 262, 264–265, 330, 332, 334, 336–337

A. shirini

Helleh subbasin /Arjan Wetland/2004 m/ N 29o 39' 20.99˝ E 51o 59' 14.39˝

Kor Basin/Maloosjan spring/1656 m/ N 29o 52' 19.7˝ E 52o 29' 48.4˝ Kor Basin/Khosroshirin springstream/2327 m/ N 30o 53' 29.5˝ E 52o 00' 36.8˝

267–273

an

A. shirini

Helleh subbasin/Arjan Wetland/2004 m/ N 29o 39' 20.99˝ E 51o 59' 14.39˝ Karun Basin/Semirom spring/1855 m/ N 31o 11' 2.3˝ E 51o 26' 59.2˝

Males

Females

13/7

20.6–29.4 (24.9±2.7)

21.1–36.0 (26.9±5.1)

9/13

22.0–27.5 (24.3±2.1)

17.5–34.0 (26.6±5.4)

3/7

24.1–26.6 (25.2±1.3)

24.3–36.0 (30.3±4.7)

24.7–35.6 (30.1±4.5) 21.2–29.6 (24.3± 3.1)

23.7–42.8 (31.7±6.5) 33.9–35.5 (34.6±0.7)

us

Comparative material A. sophiae 177–190

N ♂/♀

ip t

Collection numbers

cr

Species

6/8 4/5

Examined specimens 20 14 10 8 7 5 5 1 3 3 3 3

Sampling locality

1

GenBank Accession numbers KJ135747–KJ135766 KJ135767–KJ135780 KJ135737–KJ135746 KF559215–KF559222 KF559208–KF559214 KF910705–KF910709 KF910710–KF910714 JN565968 JX154880–82 KF910688–KF910690 JX154887–89 DQ367526, JN547802, JN547804 AF299273

1

DQ367528

Santa Pola, Spain

1

DQ981783

Southeastern Guinea

Ac ce pt e

Species/collection numbers

d

M

Table 2. GenBank accession numbers for the new specimens used in this study and the comparative material. Collections numbers are ZM-CBSU ZG303, -305–310, -325–328, -338, -122–123 for A. sophiae from the Semirom spring, which comprise seven specimens from the material indicated in Table 1 as well as seven additional specimens.

A. sophiae (Heckel, 1849) A. sophiae (Heckel, 1849) A. shirini Gholami et al., 2014 A. sophiae (Heckel, 1849) A. shirini Gholami et al., 2014 A. cf. pluristriatus (Jenkins, 1910) A. mesopotamicus Coad, 2009 A. mesopotamicus Coad, 2009 A. arakensis Teimori et al., 2012b A. farsicus Teimori et al., 2011 A. isfahanensis Hrbek et al., 2006 A. vladykovi Coad, 1988

A. fasciatus (Valenciennes, in Humboldt & Valenciennes 1821) A. iberus (Valenciennes in Cuvier & Valenciennes 1846) Epiplatys infrafasciatus (Günther, 1866)

Arjan Wetland, Iran Semirom spring, Iran Arjan Wetland, Iran Maloosjan spring, Iran Paselari spring, Iran Khonj spring, Iran Jarahi River, Iran Karkheh River, Iran Namak Lake Basin, Iran Barmeshoor spring, Iran Varzaneh spring, Iran Chaghakhor Wetland, Iran Klisova marshes, Greece

23 Page 28 of 32

ip t

Length-height-index Rel. antirostrum height Rel. rostrum height Rel. antirostrum length Rel. rostrum length Rel. medial length Rel. dorsal length Excisura angle Posterior angle Postero-ventral angle

A. sophiae (Semirom) Males, Females, N=9 N=13 12.4±0.5 12.3±1.1 11.4±0.5 11.3±0.6 14.9±1.3 14.9±1.1 5.9±0.4 5.3±0.5 9.9±1.1 10.2±0.6 8.4±0.5 8.7±0.7 26.4±1.3 27.9±1.2 15.4±1.1 15.5±1.2 ––––––– 12.3±1.3 122.2±2.4 120.9±2.1 29.7±1.0 29.2±1.4 63.3±1.5 63.5±2.0 48.9±2.4 46.8±2.0 67.0±1.9 68.3±1.9 8.9±1.5 8.4±0.9 25.3±1.3 25.4±1.8 24.2±0.7 23.7±1.7 19.8±2.3 18.5±1.6 16.3±1.4 15.9±1.5 9.0±0.5 7.8±0.7 18.8±1.4 17.3±1.3 54.4±1.0 55.0±1.4 24.2±2.1 33.9±2.1 3.21±0.28 3.16±0.34 3.75±0.21 3.68±0.37 0.93±0.03 0.94±0.04 34.2±3.8 34.1±4.2 47.0±1.7 47.3±3.4 7.2±2.3 8.6±3.9 15.5±2.7 15.8±5.0 87.5±2.8 86.0±8.4 85.3±4.1 84.5±6.9 141.0±7.9 138.1±13.1 88.8±5.2 88.6±6.7 145.0±6.2 145.5±5.8

ed

M an

A. sophiae (Arjan) Males, Females, N=13 N=7 12.5±1.1 13.0±0.8 11.3±0.9 11.1±0.9 15.8±0.9 15.9±0.9 5.5±0.5 5.7±0.8 9.8±0.8 10.1±0.7 9.2±0.7 9.4±0.5 26.8±1.6 27.6±1.1 14.9±1.1 15.1±1.2 13.1±1.4 ––––––– 119.1±3.5 117.5±1.8 28.8±1.5 28.7±1.3 60.9±2.2 60.7±0.8 47.5±6.6 45.1±3.5 67.2±1.6 67.9±2.1 8.3±1.2 8.2±0.7 28.5±3.4 27.0±2.8 24.1±1.6 25.1±1.9 19.4±2.0 18.1±1.7 17.2±1.6 15.9±1.2 8.9±1.6 8.4±0.9 17.4±1.3 16.4±0.9 54.4±4.1 53.3±1.8 34.9±1.3 33.2±1.9 2.43±0.4 2.43±0.4 2.78± 0.5 2.73±0.3 0.99±0.06 1.06±0.1 32.7±4.2 38.5±5.6 46.9±6.1 43.8±2.5 6.9±3.2 11.4±6.6 14.7±5.1 16.9±8.4 82.4±8.5 81.8±10.9 78.4±7.6 79.8±9.8 140.8±11.8 127.4±22.2 89.7±5.2 85.6±4.8 141.6±15.9 144.4±8.7

ce pt

Dorsal fin rays Anal fin rays Pectoral fin rays Pelvic fin rays Gill rakers Caudal peduncle scales Lateral scales series Predorsal scales White flank bars Total length/SL Head length/SL Predorsal distance/SL Postdorsal distance/SL Preanal distance/SL Preorbital distance/SL Maximum body depth/SL Caudal peduncle length /SL Dorsal fin depth /SL Dorsal fin length /SL Pelvic fin length /SL Pectoral fin length /SL Prepelvic distance/SL Prepectoral distance/SL Scale length/SL Scale width/SL

Ac

Otolith variables

Morphometric variables and J-indices

Meristics

Characters

us

cr

Table 3. Meristic and morphometric characters, J-indices and otolith variables for the populations of Aphanius sophiae and A. shirini (mean±standard deviation). Differences between populations of A. sophiae and A. shirini, respectively, were analyzed using ANOVA with Duncan post-hoc test for the populations of A. sophiae, and t-test for the populations of A. shirini; p<0.05; significant differences are bold-faced. N, number of specimens studied. A. sophiae (Maloosjan) Males, Females, N=6 N=8 13.3±0.8 13.8±1.2 12.7±0.4 12.1±0.6 16.0±0.9 16.0±0.9 6.0±0.0 6.1±0.4 10.8±0.9 10.9±0.6 9.8±0.8 9.0±0.8 27.8±0.8 27.6±0.9 15.7±1.4 16.1±0.6 ––––––– 14.5±1.0 120.8±1.5 118.7±1.6 30.3±1.4 29.8±1.5 62.9±1.2 62.1±1.3 43.7±1.6 42.5±1.7 67.4±1.8 68.7±1.2 7.5±0.6 7.9±0.5 33.0±2.2 30.0±1.7 22.3±1.5 22.4±1.5 21.3±1.9 16.9±1.1 18.2±1.4 16.3±0.8 9.7±1.1 8.5±0.6 18.6±0.7 16.5±1.2 53.1±1.4 54.9±1.9 30.1±4.5 34.3±1.8 4.30±0.3 4.05±0.3 5.03±0.4 4.87±0.4 1.06±0.09 1.03±0.1 35.2±5.3 37.5±5.7 45.9±4.6 45.9±3.5 7.7±3.7 9.7±5.9 15.5±4.8 15.2±5.8 82.3±8.6 82.2±6.3 79.3±6.8 81.7±8.5 134.7±15.8 132.6±19.3 84.5±8.2 87.8±7.8 142.5±11.9 141.9±3.8

A. shirini (Arjan) Males, Females, N=3 N=7 12.0±0.0 13.5±1.0 11.0±0.0 11.5±0.6 15.0±0.0 15.8±0.5 5.7±0.6 5.5±0.6 10.0±1.0 10.8±0.5 9.0±0.0 9.0±0.0 27.0±0.0 27.0±0.8 15.3±1.5 15.3±0.9 –––––––– 11.0±1.0 117.5±2.7 116.3±2.7 29.8±1.1 28.5±0.9 60.2±0.7 60.5±1.0 45.2±3.5 43.6±2.3 66.9±0.8 67.9±1.7 8.2±1.1 7.6±0.8 28.9±0.3 26.6±0.9 25.1±1.6 23.6±0.4 19.8±1.4 17.8±0.8 17.2±0.6 16.3±0.2 8.8±0.5 8.2±1.0 16.6±0.4 16.1±1.4 54.2±0.6 54.6±3.1 35.7±0.4 33.1±2.0 2.57±0.2 2.29±0.6 2.89±0.1 2.50±0.7 1.12±0.06 1.10±0.06 30.9±4.9 37.8±6.0 49.5±3.9 46.5±5.1 5.8±1.9 9.4±4.5 15.7±3.6 19.4±4.3 80.5±3.2 77.8±7.2 76.2±2.2 74.5±6.6 137.0±5.0 129.7±14.4 79.0±2.0 87.6±4.2 146.0±7.2 148.1±8.1

A. shirini (Paselari) Males, Females, N=4 N=5 10.3±0.6 10.8±0.9 10.7±0.6 10.5±0.6 15.0±0.0 15.5±0.6 6.3±0.6 5.8±0.5 10.0±1.0 9.8±0.9 10.0±1.0 10.3±0.9 28.0±1.0 28.5±0.6 20.3±1.2 21.8±0.5 ––––––– 8.3±1.2 120.5±1.5 118.0±1.8 29.6±0.1 28.2±0.9 63.2±2.1 64.7±0.4 51.2±1.1 43.8±1.6 66.9±3.7 70.5±1.6 8.4±0.5 7.3±0.6 31.1±0.6 29.4±2.2 25.2±1.2 24.1±1.2 17.3±1.6 14.9±0.9 13.3±0.5 12.5±0.5 8.7±1.4 8.3±0.2 17.5±0.5 16.8±1.2 55.2±1.3 57.3±1.9 32.9±1.2 31.6±0.9 3.8±0.5 3.4±0.3 4.6±0.4 3.8±0.2 1.11±0.12 1.25±0.06 31.2±6.4 35.0±1.6 45.4±1.7 45.7±2.8 6.2±4.4 6.7±1.6 11.3±2.8 11.2±2.5 87.0±5.2 83.9±1.5 82.4±9.8 83.1±1.8 142.3±11.9 133.3±6.5 82.3±5.0 80.0±3.6 146.67±5.7 144.3±9.7

24 Page 29 of 32

Table 4. Ranges of otolith lengths (OL, in mm) for two size-classes (SC) of the studied individuals of Aphanius sophiae and A. shirini. N, number of individuals; SD, standard deviation; SL, standard length. SC2 (20.1–27 mm SL) SC3 (27.1–35 mm SL) Species, Localities

OL

Mean±SD

OL

Mean±SD

2.35–3.39 2.58±0.28 (N= 14)

2.32–2.99 2.60±0.23 (N= 6)

A. sophiae (Semirom)

2.09–2.49 2.32±0.14 (N= 6)

2.01–2.32 2.17±0.13 (N= 5)

A. sophiae (Maloosjan)

2.43–2.81 2.59±0.16 (N=4)

2.22–2.97 2.57±0.27 (N= 7)

A. shirini (Arjan)

2.54–3.09 2.71±0.22 (N= 5)

2.33–2.80 2.62±0.22 (N= 4)

A. shirini (Paselari)

2.34–3.06 2.70±0.50 (N= 2)

2.46–2.93 2.62±0.22 (N= 4)

an

us

cr

ip t

A. sophiae (Arjan)

Ac ce pt e

d

M

Table 5. Classification matrix of the stepwise CDA (jackknifed) based on (A) meristic characters, (B) morphometric variables, (C) meristic and morphometric merged, (D) J-Indices and (E) otolith variables. The percentages in rows represent the classification into each species given in columns (correct classifications are bold-typed). Corresponding numbers of individuals are given in brackets. A, overall classification success is 67.9% (Wilk’s λ = 0.277); B, overall classification success is 73.2% (Wilk’s λ = 0.074); C, overall classification success is 85.7% (Wilk’s λ = 0.020); D, overall classification success is 91.1% (Wilk’s λ = 0.169); E, overall classification success is 48.9% (Wilk’s λ = 0.384). Species/Localities

Predicted group membership

A. sophiae (Arjan) A. sophiae (Semirom) A. sophiae (Maloosjan) A. sophiae (Arjan) A. sophiae (Semirom) A. sophiae (Maloosjan) A. sophiae (Arjan) A. sophiae (Semirom) A. sophiae (Maloosjan) A. sophiae (Arjan) A. sophiae (Semirom) A. sophiae (Maloosjan) A. sophiae (Arjan) A. sophiae (Semirom) A. sophiae (Maloosjan)

A. sophiae A. sophiae (Semirom) (Maloosjan) (A) Meristic characters 75.0% (15) 15.0 (3) 10.0 (2) 27.3 (6) 59.1% (13) 13.6 (3) 14.3 (2) 14.3 (2) 71.4% (10) (B) Morphometric variables 65.0% (13) 20.0 (4) 15.0 (3) 18.2 (4) 77.3% (17) 4.5 (1) 14.3 (2) 7.1 (1) 78.6% (11) (C) Meristic and morphometric merged 75.0% (15) 20.0 (4) 5.0 (1) 4.5 (1) 90.9% (20) 4.5 (1) 7.1 (1) 0 92.9% (13) (D) J-Indices 90.0% (18) 10.0 (2) 0 4.5 (1) 90.9% (20) 4.5 (1) 0 7.1 (1) 92.9% (13) (E) Otolith variables 35.0% (7) 25.0 (5) 40.0 (8) 23.1 (3) 69.2% (9) 7.7 (1) 28.6 (4) 21.4 (3) 50.0% (7)

NTotal

A. sophiae (Arjan)

20 22 14 20 22 14 20 22 14 20 22 14 20 13 14

25 Page 30 of 32

Appendix. Estimation of genetic divergences (Kimura 2–parameter model) between the sequences of the studied populations of Aphanius sophiae. 2

0.000 0.006 0.006 0.007 0.008 0.007 0.008

4

0.006 0.006 0.007 0.008 0.007 0.008

5

0.000 0.001 0.009 0.008 0.009

6

0.001 0.009 0.008 0.009

7

0.010 0.009 0.010

8

0.001 0.002

0.001

Ac ce pt e

d

M

an

us

cr

0.000 0.000 0.006 0.006 0.007 0.008 0.007 0.008

3

ip t

1 1. A. sophiae (Arjan) 2. A. sophiae (Arjan) 3. A. sophiae (Arjan) 4. A. sophiae (Semirom) 5. A. sophiae (Semirom) 6. A. sophiae (Semirom) 7. A. sophiae (Maloosjan) 8. A. sophiae (Maloosjan) 9. A. sophiae (Maloosjan)

26 Page 31 of 32

ip t

Fig. 1. Geographic overview showing the drainage systems of the Persian Gulf basin (dark grey shaded), the Karun River basin (light grey shaded) and the other main drainage basins in Iran. Locations of the known inland and inland-related Aphanius species in Iran are also shown. A. sophiae (Arjan) ( ), A. sophiae (Semirom) ( ), A. sophiae (Maloosjan) ( ), A. cf. pluristriatus ( ), A. mesopotamicus ( ), A. shirini (Paselari) ( ), A. farsicus ( ) A. vladykovi ( ),A. isfahanensis ( ),A. arakensis ( ), A. kavirensis ( ), A. darabensis ( ). Source of map: Coad (2014), modified.

cr

Fig. 2. The freshwater systems of the Helleh River subbasin (A) and the Karun River Basin (B). Stars indicate location of study sites. Source of maps: Google Map, modified.

an

us

Fig. 3. Maximum likelihood estimate (based on cyt b gene) of phylogenetic relationships between the populations of Aphanius sophiae and A. shirini and other inland and inlandrelated Aphanius species in Iran. Numbers above nodes represent maximum likelihood bootstrap values based on 2,000 replicates followed by Bayesian likelihood values. Species and locations correspond to those listed in the Material section. 1n(L)= –5016.3207, α =0.2920. Scale bar indicates substitutions per site.

d

M

Fig. 4. Males (left) and females (right) of the studied species and populations of Aphanius from the Zagros Mountains. Photos of A. sophiae (Maloosjan) and A. shirini (Paselari) have been used previously in Gholami et al. (2014).

Ac ce p

te

Fig. 5. SEM images of left otoliths (medial view) of the studied species and populations of Aphanius. The otoliths represent three males (left) and three females (right) in each group. The otoliths of A. sophiae (Maloosjan) and A. shirini (Paselari) have been used previously in Gholami et al. (2014). Fig. 6. A Main tectonic structures of the Zagros Mountains; frame indicates study area shown in B; B Close-up of study area showing details of the Kazerun Fault System with the Dena, Kazerun, Kareh Bas, Sabz Pushan, Sarvestan and Borazjan faults and locations of A. sophiae (Arjan) ( ), A. sophiae (Semirom) ( ), A. sophiae (Maloosjan) ( ), A. cf. pluristriatus ( ), A. shirini (Paselari) ( ), and A. farsicus ( ). HZF, High Zagros Fault; KFS, Kazerun Fault System; MRF, Main Recent Fault; MZRF, Main Zagros Reverse Fault; MFF, Main Frontal Fault; ZFF, Zagros Frontal Fault. Source of maps: Hatzfeld et al. (2010), modified.

Page 32 of 32